Summary :The aim of our study was to detect free-living Amoebae (FLA) by morphological methods and to identify Acanthamoeba spp. by PCR in the dental unit water lines (DUWL). Materials and methods: it was a prospective study dealing with 196 water samples collected from DUWL (94 samples taken in the early morning before materials flush and patient consultations and 102 samples taken after consultations). At the same time, 39 samples from tap water were realized. Results: 135 (69 %) samples were positives by the morphological study with morphotypical diversity. The predominant morphotype was the monopodial (39.2 %). 18 strains of Acanthamoeba spp. were detected in DUW (13.3 %) and three strains in tap water (10 %). The amplification of 18S rDNA gene of these strains of Acanthamoeba spp. was positive for all samples. Conclusion: the FLA and Acanthamoeba were isolated both in tap water and in dental unit. The amoeba pathogenicity has not been demonstrated after oral or dental contamination; but the presence of intracellular and pathogenic bacteria in the amoeba could be a source of microbiological risks for patients in case of deep dental care or immunodepression. The improvement of this dental unit was necessary by putting a filter of 0.2 microns porosity before the arrival of the water in hand-pieces allowing the limitation of FLA passage. Résumé